The goal of the experiments described in this proposal is to develop a novel unbiased, genome-wide assay to identify the direct mRNA targets of microRNAs (miRNAs). Identification of target gene mRNAs is critical for elucidating the role of miRNAs in development and disease, yet no assay exists for experimentally identifying direct interactions between miRNAs and candidate target mRNAs. We propose to address this gap in knowledge by developing an assay that combines RNA immunoprecipitation (RIP) with a novel method we call sequencing of isolated RNAs (SIR). As a proof of principle application we will develop our RIP-SIR assay in the context of identifying changes in miRNA expression and target affinities that characterize the transition from early-stage to late stage pulmonary adenocarcinomas in genetically engineered mice. Our approach involves three steps: 1) profiling expression of miRNAs and target mRNAs; 2) determining which miRNAs and mRNAs are differentially expressed; and 3) identifying which miRNAs are bound to individual mRNA targets. The RIP-SIR assay will provide the basis for further development of microRNAs as potential diagnostic, prognostic, and therapeutic targets for treatment of lung cancer in humans. ) PUBLIC HEALTH RELEVANCE: MicroRNAs (miRNAs) play a central role in regulating fundamental biological processes such as cell proliferation, differentiation, apoptosis and metastasis and, therefore, are potentially valuable targets for novel therapies. Initiation of clinical trials for miRNA-based therapies, however, requires the ability to accurately identify which transcripts will be targeted by individual miRNAs. The goal of this proposal is to develop a novel, genome-wide assay to identify changes in microRNA (miRNA) expression and target affinity that define the transition from early-stage to late-stage pulmonary adenocarcinoma in genetically engineered mice. Successful completion of this proposal will provide the basis for further development of miRNAs as potential diagnostic, prognostic and therapeutic targets for treatment of lung cancer in humans. Although we are developing this assay to study the role of miRNAs in lung cancer progression, this assay is applicable to study any of the diverse cellular processes and disease states in which gene expression is presumably regulated by miRNAs.